ABSTRACT
Exendin-4 is a GLP 1 agonist incretin-mimetic peptide hormone comprising 39 amino acids. Exenatide (Byetta®) is a chemically synthesized version of Exendin-4 with an additional C-terminal amidation. Exenatide acts as a GLP-1 receptor agonist. This paper illustrates the method adopted for cloning, fermentation and purification of recombinant Exendin-4 analog expressed in Escherichia coli. The biologically expressed analog was extensively characterized using different orthogonal methods to confirm their biological activity and physicochemical properties. It was observed that the expressed analog showed comparable functional properties as that of Byetta® irrespective of their modes of development. Further, in vivo efficacy of the recombinant Exendin-4 analog was studied in Oral Glucose Tolerance Test (OGTT) in mice models. Byetta® and Exendin-4 analog treated groups showed comparable glucose lowering activity in the OGTT model.
Subject(s)
Escherichia coli , Gene Expression , Peptides , Venoms , Animals , Drug Evaluation, Preclinical , Exenatide , Male , Mice , Peptides/genetics , Peptides/isolation & purification , Peptides/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Venoms/biosynthesis , Venoms/genetics , Venoms/isolation & purification , Venoms/pharmacologyABSTRACT
Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNA-unwinding element-binding protein DUE-B in vivo. We present here the 2.0A crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.